Cardiac arrhythmias, including atrial fibrillation, are a high risk factor for stroke and sudden cardiac death. Cardiac alternans is a key arrhythmogenic factor predisposing the heart to re-entry and lethal arrhythmias. At the cellular level, alternans occurs as electrical (action potential duration, APD), mechanical and Ca alternans. In atrial cells Ca release during excitation-contraction coupling is spatially inhomogeneous. Furthermore, Ca alternans in atrial cells reveals subcellular gradients and subcellular regions alternating out-of-phase, leading to a high propensity of spontaneous pro-arrhythmic Ca release and Ca waves. Membrane voltage (Vm) and [Ca]i regulation are bidirectionally coupled, such that instabilities in Vm (APD restitution properties) and intracellular Ca cycling dynamics result in electromechanical and Ca alternans. The mode of coupling determines the form of alternans and severity of arrhythmogenesis. The overall goal of this proposal is to test experimentally at the single cell level how bidirectional coupling between Vm and [Ca]i leads to APD and Ca alternans and generates conditions that favor atrial arrhythmia. The following specific aims are proposed: Specific aim 1: determine how instabilities in Vm affect [Ca]i and lead to alternans (Vm?[Ca]i coupling). Hypothesis: impaired APD restitution in atrial myocytes leads to pacing-induced APD and Ca alternans. Specific aim 2: determine how instabilities in Ca cycling affect Vm and lead to alternans ([Ca]i ?Vm coupling). Hypothesis: the interdependence of 2 key parameters of beat-to-beat Ca cycling - (1) the non-linear relationship between SR Ca release and SR load (fractional release) and (2) the efficiency of Ca sequestration (cytosolic Ca buffering, Ca sequestration by SERCA, mitochondrial Ca buffering, Ca extrusion) - lead to APD and Ca alternans. Specific aim #3: determine how the restitution properties of Ca release (global Ca transient and elementary Ca sparks) are related to alternans. Hypothesis: under conditions that favor [Ca]i -driven alternans the restitution of elementary Ca release events is slowed which favors Ca alternans. To achieve these aims a multitude of experimental techniques will be used: high resolution imaging by laser scanning confocal microscopy in single atrial myocytes to measure whole cell and subcellular [Ca]i, [Ca]mito and [Ca]SR, whole-cell voltage and current clamp techniques to study membrane currents and Vm (APD), subcellular photolysis of caged Ca, adenoviral gene-transfer, and pharmacological manipulation of Ca transport and buffering. Experiments will be conducted on adult cat atrial myocytes and atrial myocytes isolated from transgenic mice with altered expression of Ca handling proteins (calsequestrin, phospholamban). The proposed research will provide fundamental new information on the cellular mechanism of APD and Ca alternans relevant to the understanding of atrial arrhythmias. PUBLIC HEALTH RELEVANCE: Cardiac alternans (T-wave alternans, pulsus alternans) has been recognized as a risk factor for cardiac arrhythmia and sudden cardiac death, and has been linked directly to atrial fibrillation, the most common form of cardiac arrhythmia. At the cellular level electrical (action potential duration) and intracellular calcium alternans correlate with arrhythmogenic calcium release. This study seeks to determine in atrial myocytes, at the cellular and subcellular level, the mechanisms of disturbances of electrical activity and calcium regulation that favor the occurrence of alternans and atrial arrhythmia.